Detection of Listeria monocytogenes in Milk and Ice Cream by Polymerase Chain Reaction

Abstract

Abstract

The present study was undertaken to standardize PCR assay for detection of L. monocytogenes and Listeriolysin O from livestock foods and compare its efficacy with conventional cultural methods. A set of primer derived from iap gene and other set derived from hlyA gene were used for detection of L. monocytogenes and Listeriolysin O in the PCR assay. The PCR assay was initially standardized by optimizing the concentration of the components of the reaction mixture and by annealing temperatures and cycling conditions for two sets of primers. Electrophoresis analysis revealed the specific amplification products at 131 and 456 bp respectively for iap and hlyA genes. Three different template preparation methods, viz., genomic DNA extraction, heat lysis and lysis buffer methods were compared to determine the most sensitive, rapid and simple method suitable for PCR technique. In this study, heat lysis method was employed as it was simple, rapid, reliable and the efficiency was comparable to genomic DNA extraction method. The specificity of the standardized PCR assay for the two primers was tested by subjecting eight isolates including L. monocytogenes and seven other non-Listeria monocytogenes bacteria. Only L. monocytogenes isolates gave specific product of 131 bp for iap and 456 bp for hlyA genes respectively. The sensitivity of the PCR assay was evaluated by subjecting serial 10-fold dilution of pure culture of L. monocytogenes from 4.0 × 10⁷ to 4.0 cfu/mL to PCR assay with two sets of primers. The minimum detection level was found to be 4 cfu/mL. Four different broths, i.e., two non-selective and two selective broths were evaluated to assess their PCR compatibility using four different treatments for concentrating the target organism. Two non-selective broths (BHI and TSB) produced bands in all four treatments, but they were found as light. Among selective broths, LEB gave very bright bands to treatment-1 and 2 and bright bands to other treatments whereas PALCAM medium gave bright bands to treatment-1 and 2, and gave light bands to other two treatments.

Keywords: Listeria monocytogenes, Listeriolysin O, milk, ice cream, PCR

Cite this Article:
Swetha CS, Jagadeesh Babu A, Madhava Rao T, et al. Detection of Listeria monocytogenes in milk and ice cream by polymerase chain reaction. Research and Reviews: Journal of Microbiology and Virology. 2015; 5(1): 24–35p.